The ultimate goal of this project is to integrate microfluidic devices with NASA's space bioreactor systems. In such a system, the microfluidic device would provide realtime feedback control of the bioreactor by monitoring pH, glucose, and lactate levels in the cell media; and would provide an analytical capability to the bioreactor in exterrestrial environments for monitoring bioengineered cell products and health changes in cells due to environmental stressors. Such integrated systems could be used as biosentinels both in space and on planet surfaces. The objective is to demonstrate the ability of microfabricated devices to repeatedly and reproducibly perform bead cytometry experiments in micro, lunar, martian, and hypergravity (1.8g)

Culbertson, Christopher

Gonda, Steve

Ramsey, John Michael

NASA Technical Reports Server

 9 
 2.2 Experimental Microfluidic System 
FLIGHT DATES: 
June 28 - July 2, 2004 
PRINCIPAL INVESTIGATORS: 
Christopher Culbertson, Kansas State University 
Steve Gonda, NASA/Johnson Space Center 
John Michael Ramsey, University of North Carolina 
 
 
 
GOAL: 
The ultimate goal of this project is to integrate microfluidic devices with NASA’s space 
bioreactor systems. In such a system, the microfluidic device would provide realtime feedback 
control of the bioreactor by monitoring pH, glucose, and lactate levels in the cell media; and 
would provide an analytical capability to the bioreactor in exterrestrial environments for 
monitoring bioengineered cell products and health changes in cells due to environmental 
stressors. Such integrated systems could be used as biosentinels both in space and on planet 
surfaces.  
OBJECTIVE: 
To demonstrate the ability of microfabricated devices to repeatedly and reproducibly perform 
bead cytometry experiments in µ, lunar, martian, and hypergravity (1.8g).  
https://ntrs.nasa.gov/search.jsp?R=20050217273 2019-08-29T19:34:05+00:00Z
 10 
 BACKGROUND:  
Microfluidic or Lab-on-a-Chip devices are small platforms upon which a complete chemical 
analysis can be performed. These devices consist of a series of small interconnecting channels 
(10µm deep and 40 to 200mm wide) etched in glass, or molded in polymers, through which 
fluids can be moved. The fluids can either be controlled with electric potentials generating 
electric fields within the channels that move fluid electrokinetically, or by generating pressure 
differentials using a syringe pump or peristaltic pump that moves the fluid hydrodynamically. 
Using these two methods of controlling fluid flow it is possible to generate devices that have 
multiple uses. For example it is possible to use hydrodynamic flow to rapidly move large 
numbers of cells or large particles, such as beads, through a focusing intersection much faster 
than if one were to use electrokinetic flow. On the other hand electric fields allow for the 
separation of differentially charged analytes using capillary electrophoresis, something 
hydrodynamic flow alone can not accomplish. These devices provide many advantages over 
conventional bench top scale instrumentation as a result of their ability to integrate sample 
handling and sample processing operations with analyte detection on a single, monolithic 
substrate. Such integration allows for the efficient automation of chemical analyses. In addition 
to automation and integration, microchips have several other inherent advantages over 
conventional chemical analysis instrumentation. These advantages include (1) the ability to 
perform faster separations with no loss in separation efficiency, (2) lower reagent and sample 
consumption (< 1mL/year), (3) less waste production, and (4) the ability to fabricate many 
parallel systems on the same device. Thus far, their performance has been either equivalent to or 
better than conventional laboratory devices in all cases investigated. They appear to offer the rare 
combination of better-faster-cheaper simultaneously, and their ability to manipulate reagents and 
reaction products “on-chip” suggests the potential to perform virtually any type of “wet-
chemical” bench procedure on a microfabricated device.  
The advantages described above make these devices especially interesting for use in 
extraterrestrial environments where small, portable, rugged, and reliable devices capable of 
sustained remote automated operation will be required.  
 11 
 METHODS AND MATERIALS: 
Microfluidic Experiment Description 
The portable microfluidic device developed for these tests contained in a Bud box enclosure 
(NBA10148) which had exterior dimension of approximately 30 cm wide x 18 cm deep x 40 cm 
high. The microchips, in their custom machined 2-part PMMA holder, were attached to an x-y 
positioning plate (ST1XY-S; Thor Labs Inc.; Newton, NJ) and positioned above a microscope 
objective (CD-240-M40X; creative devices, Neshanic Station, NJ). This objective was used to 
focus the excitation light of a green laser pointer (The Laser Guy, Seabrook, TX). The laser was 
modified to have a remote switch and power supply (2 D cell batteries). The laser module 
consisted of an 808 nm laser diode which was shifted to 1064 nm and then frequency doubled to 
provide a continuous output of 5 mW at 532 nm. The laser beam was reflected off a dichroic 
mirror (560 DRLP: Omega Optical, Brattleboro, VT) prior to being focused into the microchip 
channel by the microscope objective. The fluorescence from the labeled amino acids was 
collected by the same microscope objective, passed through the dichroic mirror, a 1.0 mm 
pinhole, and a 565 nm longpass filter (565ALP, Omega Optical) prior to being detected at a 
channel photomultiplier tube (MD972; Perkin Elmer; Fremont, CA). The PMT was powered by a 
5 volt power supply. The gain was manually controlled by a potentiometer which had a locking 
mechanism to prevent accidental change.  
The high voltages used for making injections and performing the electrophoretic separations on 
the microchip were provided by two independent high voltage power supplies cabable of 125 ma 
outputs at up to 8 kv (C80; Emco High Voltage Corp.). Each high voltage power supply was 
powered by a 15 VDC source. The HV output was determined by a 0-5 VDC control signal 
provided by a National Instruments AO card (DAQCard AO-2DC). These power supplies each 
occupied only 19 cm3 and weighed 51 g, making them very suitable for portable applications.  
A syringe with a locking stop was used to provide pressure to drive the hydrodynamic flow for 
the bead and cell focusing experiments.  
The entire instrument was controlled and data were acquired using in-house written LabVIEW 
software run off a laptop computer.  
Protocol 
Orange fluorescent polystyrene microspheres that have a 10µm diameter were diluted into the 
running buffer to make a bead concentration of 6.5x106 beads/ ml (Molecular Probes, Eugene, 
OR). The run buffer was composed of 25 mm NaBorate and 0.01% (v/v) Silwet. The Silwet 
helped prevent aggregation of the particles and particles adsorption to the capillary sidewalls. 
Sonication of this solution was performed prior to focusing experiments to further reduce bead 
aggregation.  
To perform the bead focusing experiments a 25 mm NaBorate and 0.01% Silwet solution were 
placed in channels B and C (Figure 1), while the bead solution was placed in channel A. A low 
pressure was applied to channel D using a syringe pump. This caused fluids from A, B, and C to 
converge at the cross intersection. The beads were consequently focused and detected using the 
LIF system described above.  
 12 
 
 
Figure 1: Schematic of bead cytometry experiment, and CCD imaging of 10 micron spheres being 
focused at the cross intersection. 
RESULTS: 
Bead cytometry was performed on two of the three flights, June 30th and July 1st. Figure 2 below 
shows an example of the bead cytometry experiment at 0.026 g. There was no significant 
difference between the cytometry experiment at 0.026 g, and 1.768 g. The particle velocity was 
2.3 meters per second and the volumetric flow rate was 0.036ml/s. To properly sample the 
rapidly flowing beads, the data acquisition rate used was 20 kHz. The maximum particle count 
rate at this data acquisition rate is about 3,000/s. Our experiment had a count rate of 200/s with 
an average intensity of 0.95±0.4. The velocity of the beads, maximum count rate, average and 
standard deviation of the intensity and flow rate can be seen in Table 1.  
 
 
 
 
 
 
 
A 
B 
 
C 
D 
A 
B 
D 
C 
 13 
 
1.0
0.8
0.6
0.4
0.2
0.0
Fl
u
or
es
ce
n
ce
 (A
rb
.
 
Un
its
)
6.526.516.506.496.486.47
Time (s)
 
Figure 2: 10 seconds of bead cytometry at 0.026 g 
0.8
0.6
0.4
0.2
0.0
Fl
uo
re
sc
en
ce
 (A
rb
. U
n
its
)
6.48466.48456.48446.48436.48426.4841
Time (s)
 
 14 
 The final flight on July 2nd was used to attempt to complete an experiment integrating a 
bioreactor with a microfluidic device to perform an on-line separation of amino acids within the 
bioreactor. The separation was working on the ground prior to the flight, but unfortunately an 
electrical failure with the laser catastrophically caused the experiment to fail. This problem was 
solved by integrating a new 30 mW laser that has a longer lifetime for future flights. 
DISCUSSION: 
This data shows that it is possible to perform bead cytometry in reduced gravity environments. 
The throughput of the device is absolutely outstanding with respect to the number of beads it can 
count per second, although the standard deviation of the mean amplitude of the fluorescent signal 
is relatively high. This suggests that the focusing at the intersection needs to be tighter to assure 
that the particles pass through the interrogation beam single file. 
Table 1: Summary of Results for Bead Cytometry Experiments 
Bead Velocity 2.3 m/s 
Max. Count Rate 3,000 bead/s 
Fluidic Flow Rate 0.036 mL/s 
Average Peak 
Amplitude 0.9+/- 0.4 
PHOTOGRAPHS: 
JSC2004E28082 to JSC2004E28085 
JSC2004E28254 
JSC2004E28286 
JSC2004E28399 to JSC2004E28401 
VIDEO: 
• Zero g  June 29 – July 2, 2004, Reference Master: 718394 
Videos available from Imagery and Publications Office (GS4), NASA/JSC. 
CONTACT INFORMATION: 
Christopher Culbertson, Ph.D. 
Kansas State University 
Phone: 785-532-6685 
Fax: 785-532-6666 
culbert@ksu.edu 


Experimental Microfluidic System

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